Adenosine can attenuate traumatic and ischemic cell death, arrest seizure activity, and precondition neurons against subsequent injury. The cells, molecular mechanisms, and subtypes of adenosine receptors and G proteins that are involved in these responses are incompletely understood. The purpose of this research is to investigate the structure, function and CNS cellular location of adenosine receptor (AR) subtypes. In order to do this a novel expression plasmid has been constructed and used to express all four recombinant human ARs (A1, A2A, A2B, and A3) in mammalian or insect cells. In addition to expressing wildtype receptor, the four ARs have been double-tagged by extending their amino termini with hexahistidine (H) and the FLAG epitope (F) to make H/F-ARs. A highly efficient purification scheme has been developed that utilizes sequential anti-FLAG antibody columns and Nitrilotricetic acid columns to purify to homogeneity functional ARs in high yield. The goal of this proposal is to learn more about the pharmacology, ligand binding domains, G protein coupling, phosphorylation and CNS cellular location of the various adenosine receptor subtypes. There are four specific aims. Aim 1 is to investigate the pharmacology of recombinant human AR subtypes, particularly A2B and A3 that have been poorly characterized to date. Aim 2 is to investigate interactions between purified recombinant human A1AR and purified G proteins of defined subunit composition produced in the laboratory of a collaborator, Dr. James Garrison. The applicants also will determine if desensitization alters the amount or composition of G proteins that can be co-purified with H/F-A1AR. Aim 3 is to use peptide sequencing to identify particular amino acids that are covalently modified on recombinant ARs following phosphorylation or photoaffinity labeling. Aim 4 is to investigate the cellular and subcellular distribution of A1, and A2A AR in the rat CNS by immunocytochemistry. They have shown that purified human H/F-A2AAR injected into mice produce antibodies useful for immunocytochemistry. This research will increase our understanding of the structure and function of AR and may contribute to the development of new therapies to treat epilepsy, stroke and SlDS.